Nicotinic attenuation of cns inflammation and autoimmunity

ABSTRACT

The present invention relates to methods of treating and/or ameliorating the severity of inflammation and autoimmunity in the central nervous system (CNS). In one embodiment, the present invention provides a method of treating multiple sclerosis by administering a therapeutically effective dosage of nicotine, or a pharmaceutical equivalent, analog, derivative, or salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/451,318, filed Aug. 4, 2014, which is adivisional application of U.S. Pat. No. 8,841,329, filed May 5, 2011,which is the National Phase of International Application PCT/US09/56671,filed Sep. 11, 2009, which designated the United States and waspublished under PCT Article 21(2) in English, and which further includesa claim of priority under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. No. 61/096,170, filed Sep. 11, 2008. The contents ofall the related applications cross-referenced herein are herebyincorporated by reference in their entirety as through fully set forthherein.

GOVERNMENT RIGHTS

This invention was made with Government support under Grant No. AI052463awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Previous studies have suggested that nicotine, an endogenousneurotransmitter and psychoactive component of cigarette smoking, hasprofound immunological effects (McAllister-Sistilli, C. G., et al.,Psychoneuroendocrinology 23:175-187; Sopori, M., Nat Rev Immunol2:372-377). During ontogeny, nicotine elevates expression of therecombinase-activating gene on developing thymocytes in the thymus(Middlebrook, A. J., et al., J Immunol 169:2915-2924) and regulates Bcell development in the bone marrow (Skok, M. V., et al., Life Sci80:2334-2336). For mature lymphocytes, nicotine suppresses the T cellresponse and alters the differentiation, phenotype and functions ofantigen-presenting cells (APCs) including dendritic cells(Nouri-Shirazi, M., et al., Immunol Lett 109:155-164; Guinet, E., etal., Immunol Lett 95:45-55) and macrophages (Floto, R. A., et al.,Lancet 361:1069-1070).

The impact of nicotine on immune responses in vivo, however, isextremely complex depending on the dosage, the duration of exposure aswell as the involvement of specific organ systems in which immuneresponses evolve. Nicotine dampens inflammation and reduces mortality ina mouse model of sepsis (Wang, H., H. et al., Nat Med 10:1216-1221).This compound additionally reduces the incidence of type 1 diabetes inmice (Mabley, J. G., et al., J Pharmacol Exp Ther 300:876-881) andalters humoral autoimmunity in the experimental model, systemic lupuserythematosus (SLE) of mice (Rubin, R. L., et al., Toxicol Sci87:86-96). Several epidemiological studies reveal a strong inversecorrelation between smoking and the autoimmune response, the clinicalmanifestations of SLE and ulcerative colitis (Rubin, R. L., et al.,Toxicol Sci 87:86-96; Jani, N., et al., Gastroenterol Clin North Am31:147-166). Other studies suggest that smoking might be associated withthe exacerbation of multiple sclerosis (MS) and Crohn's disease (Emre,M., et al., Arch Neurol 49:1243-124; Friend, K. B., et al., DisabilRehabil 28:1135-1141; Johnson, G. J., et al., Aliment Pharmacol Ther21:921-931). Confounding factors conferring disease exacerbation, aswell as dosage and duration of smoking in these studies likelycontribute to the discrepancy observed between the possible positive vs.negative effects of nicotine.

Inflammatory and immune responses within the central nervous system(CNS) are capable of shaping the clinical outcome of CNS diseasesincluding stroke, trauma, Alzheimer's disease, Parkinson's disease,epilepsy, encephalomyelitis and MS (Zipp, F., et al., Trends Neurosci29:518-527). Compared to other organ systems, the CNS has several uniqueproperties with respect to immune responses. First, the spectrum of APCsdiffers from that in the periphery, because in the CNS, residentmicroglia and astrocytes are active participants (Ponomarev, E. D., etal., J Immunol 178:39-48; Simard, A. R., et al., Mol Psychiatry11:327-335). Second, cells from the periphery that migrate into the CNSencounter myelin and other antigens, then undergo reactivation enhancingtheir capacity to recognize a wide spectrum of ambient antigens, aprocess defined as determinant spreading (McMahon, E. J., et al., NatMed 11:335-339). Third, given the physical proximity of neuronal cells,the nature and magnitude of immune responses within the CNS are likelyinfluenced by signals stemming directly from the local environment.Despite the quite extensive literature on the impact of nicotine onimmune responses in various organ systems, the influence of nicotine onCNS inflammation has not been investigated.

Accordingly, there is a need in the art for greater understanding of therole of nicotine in CNS inflammation, as well as a need to develop novelmethods of treatment for conditions associated with CNS inflammation andautoimmunity.

SUMMARY OF THE INVENTION

Various embodiments include a method of treating a disease or conditionin a subject, comprising providing a composition comprising a nicotinicreceptor agent, and administering a therapeutically effective dosage ofthe composition to the subject. In another embodiment, the nicotinicreceptor is an α7 nicotinic acetylcholine receptor. In anotherembodiment, the subject is a mouse. In another embodiment, thetherapeutically effective dosage of the nicotinic receptor agent rangesfrom 1 mg nicotine free base/kg to 60 mg nicotine free base/kg. Inanother embodiment, the therapeutically effective dosage of thenicotinic receptor agent is about 13 mg nicotine free base/kg/dayadministered over a period of 7 days. In another embodiment, the subjectis a human. In another embodiment, the therapeutically effective dosageof the nicotinic receptor agent ranges from nicotine plasma levels of 1ng/mL to 100 ng/mL. In another embodiment, the therapeutically effectivedosage of the nicotinic receptor agent is a nicotine plasma level ofabout 30 ng/mL. In another embodiment, the therapeutically effectivedosage of the nicotinic receptor agent ranges from 0.1 mg to 5 mg. Otherembodiments include the therapeutically effective dosage of thenicotinic receptor agent as about 1 mg. In other embodiments, thetherapeutically effective dosage of the nicotinic receptor agent rangesfrom 0.015 mg/kg/day to 0.6 mg/kg/day administered over a period of 7days. In another embodiment, the disease or condition is multiplesclerosis (MS). In another embodiment, the disease or condition is acutedisseminated encephalomyelitis. In another embodiment, the disease orcondition is experimental autoimmune encephalomyelitis (EAE). In anotherembodiment, the disease or condition is a neuroimmunological disease. Inanother embodiment, the nicotinic receptor agent is administered to thesubject continuously by an implanted pump. In another embodiment, thenicotinic receptor agent is administered by direct injection to thesubject. In another embodiment, the nicotinic receptor agent comprisesnicotine, or a pharmaceutical equivalent, analog, derivative, or saltthereof. In another embodiment, the nicotinic receptor agent comprisesone or more substances listed in Table 1. In another embodiment, thenicotinic receptor agent comprises nicotine bitartrate.

Other embodiments include a method of treating a disease in a subject,comprising providing a composition comprising a nicotinic receptoragent, and administering a therapeutically effective dosage of thecomposition to enhance the activity of a compound that treats thedisease. In another embodiment, the disease is MS. In anotherembodiment, the disease is a neuroimmunological disease.

Other embodiments include a method for the treatment of a disease and anassociated condition, comprising administering a first composition in anamount effective to treat the disease, administering a therapeuticallyeffective dosage of a second composition comprising a nicotinic receptoragent to treat the associated condition. In another embodiment, thedisease is MS. In another embodiment, the disease is aneuroimmunological disease. In another embodiment, the associatedcondition is inflammation. In another embodiment, the associatedcondition is an autoimmune effect.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIGS. 1A-1D depict nicotinic attenuation of actively induced andadoptively transferred EAE. Nicotine or PBS were administered at a doseof 13 mg/kg daily for a total of 7 doses, and treatment started at theindicated days (−7, FIG. 1A; 0, FIG. 1B; +7, FIG. 1C). The mice wereobserved daily and scored for clinical disease. Data represent mean±SDof total mice in each group (10 to 12 mice/group). Statisticalevaluation was performed to compare experimental groups andcorresponding control groups. Results for adoptively transferred EAE areillustrated in FIG. 1D (n=5 each group).

FIGS. 2A-2G depict nicotine ameliorates CNS inflammation, demyelinationand axonal damage. At day 25 p.i., mice were sacrificed, and CNS tissueswere fixed in formalin then processed for staining with H&E (leftpanel), LFB (middle panel) or Biechowsky silver (right panel). FIGS.2A-2C, control group. FIGS. 2D-2F, nicotine-treated group. Originalphotos were taken at 200×. FIG. 2G, Seminal quantitative summary ofinflammation, demyelination and axonal damage. To calculate the extentof CNS pathology, the percentages of spinal cord inflammation,demyelination and axonal damage per mouse were calculated by firstdetermining the total white matter area for all spinal cord sections bymanually tracing the regions. Next the areas of spinal cordinflammation, demyelination and axonal damage were determined bymanually tracing each section. Pathological changes of each spinal cordwere scored as described herein. n=3 for all experiments. *P<0.05.Mann-Whitney U-test was used for the comparison.

FIGS. 3A-3D depict nicotine alters the peripheral lymphocytesubpopulation during EAE. On the day of immunization to incite acuteEAE, mice were treated with nicotine at a dose of 13 mg/kg or PBS dailyfor a total of 7 doses. Mice were killed on day 11 after immunization,and mononuclear cells were isolated from their spleens as describedherein. The dot plots generated after gating on lymphocytes (by forwardvs. side scatter) are shown for T cells and B cells. FIGS. 3A-3C,Representative dot plot results for CD4⁺, CD8⁺, NK, NKT cells, and CD3⁻CD19⁺ cells. FIG. 3D, The absolute number of CD4⁺, CD8⁺, CD3⁺, and CD3⁻CD19⁺ cells. (n=5 mice each group). Data presented in FIGS. 4-8 werealso generated using cells prepared at this time point.

FIGS. 4A-4C depict nicotine augments the expression of FoxP3 onregulatory T cells. FIG. 4A, Representative plots from individual miceshowing the percentage of CD4⁺CD25⁺ T cells gated on lymphocytes (n=5).FIG. 4B, The expression of Foxp3 in relation to CD25⁺ cells wasdetermined by gating on CD4⁺ cells (n=5). FIG. 4C, The averagepercentage of CD4⁺CD25⁺ T cells and Treg⁺/FoxP3⁺ cells in PBS- andnicotine-treated mice. The average percentage of Foxp3 and CD25 doublepositive cells was calculated by gating on CD4⁺ cells in the PBS- andnicotine-treated mice.

FIGS. 5A-5D depict nicotine reduces the expression of MHC II, CD80, andCD86 on APCs in the periphery. FIGS. 5A and 5C, MHC II, CD80 and CD86expression was analyzed with gating on macrophages (CD11b⁺)(representative histogram plots ([[A]] FIG. 5A); absolute numbers ([[C]]FIG. 5C)). FIGS. 5B and 5D, MHC II, CD80, and CD86 expression wasanalyzed with gating on dendritic cells (CD11c⁺) in the periphery(representative histogram plots (FIG. 5B); absolute numbers (FIG. 5D)).(n=5 mice each group). (*, p<0.05 vs. PBS, **, p<0.01 vs. PBS).

FIGS. 6A-6C depict nicotine inhibits peripheral autoreactive T cellresponses. FIG. 6A, The proliferation of antigen-specific splenic cellsfrom PBS- or nicotine-treated mice was measured as ³H incorporation bythese cells cultured with PLP or MOG. Results are expressed as meancpm±SD. FIG. 6B, Proliferation was assessed after CFSE staining of CD3⁺T cells, CD4⁺ T cells, and CD8⁺ T cells. FIG. 6C, Splenic cell apoptosisand death were detected by annexin V and PI double staining.Representative results from one of three independent experiments areshown (n=5 mice each group). (*, p<0.05 vs. PBS, **, p<0.01 vs. PBS).

FIGS. 7A-7F depict effects of nicotine on the Th response in EAE mice. Tcells were isolated from the groups of mice specified in FIG. 3 andcultured in the presence of MOG and other stimuli. Supernatants wereharvested after 36 h, and IFN-γ (FIG. 7A), IL-2 (FIG. 7D), IL-10 (FIG.7E) and TGF-β1 (FIG. 7F) production was determined by ELISA. FIGS. 7Band 7C, IFN-γ secretion upon stimulation with MOG in nicotine- andPBS-treated mice was quantified by intracellular cytokine staining. FIG.7B, Representative results appear in dot plots for CD4, CD8 and IFN-γ.FIG. 7C, The average percentage of CD4⁺ IFN-γ⁺ cells and CD8⁺ IFN-γ⁺cells is shown. The data are means±SD of values from 9 mice per group.

FIG. 8 depicts nicotine alters autoantibody isotypes. Sera werecollected on day 11 after immunization, and the titers of MOG-specificIgG (1:500), IgG1 (1:500), IgG2b (1:500), IgG3 (1:100), IgA (1:100) andIgG2a (1:2) were determined by ELISA. Data are means±SD from three miceper group. Similar results were obtained in two independent experiments.(*, p<0.05 vs. PBS, **, p<0.01 vs. PBS).

FIGS. 9A and 9B depict nicotine alters lymphocyte subpopulations in theCNS during EAE. On the day of immunization to incite acute EAE, micewere treated with nicotine at a dose of 13 mg/kg or PBS daily for atotal of 7 doses. Mice were killed on day 11 after immunization, andmononuclear cells were isolated from the CNS. The dot plots generatedafter gating on mononuclear cells (by forward vs. side scatter) areshown for T cells and B cells. FIG. 9A, Representative dot plots areshown for CD3⁻ CD19⁺ cells (upper panel) and CD4⁺ CD8^(°) cells (lowerpanel). FIG. 9B, The Absolute numbers appear for CD3⁺, CD3⁻CD19⁺, CD4⁺,and CD8⁺ cells.

FIGS. 10A-10F depict nicotine inhibits activation-induced MHC class IIexpression and suppresses co-stimulatory molecules in APCs. Themononuclear cells were isolated from CNS as in FIGS. 9A and 9B. FIG.10A, The representative dot plot recapitulates results for dendriticcells (CD11b⁺CD11c⁺, upper panel) and co-stimulators (CD80⁺/CD86⁺, lowerpanel) on mononuclear cells. FIG. 10B, The absolute numbers of dendriticcells (CD11b⁺CD11c⁺) and co-stimulators (CD80⁺/CD86⁺) are also shown onmononuclear cells. FIGS. 10C and 10D, MHC II, CD80 and, CD86 expressionwas analyzed by gating on live macrophages (CD11b⁺) in the CNS (C arethe representative histogram plots; D are the absolute numbers). FIGS.10E and 10F, MHC II, CD80 and, CD86 expression was analyzed by gating onlive dendritic cells (CD11c⁺) in the CNS (E are the representativehistogram plots; F are the absolute numbers). (*, p<0.05 vs. PBS, **,p<0.01 vs. PBS).

FIG. 11 (prior art) depicts an example of a signaling pathway of the α7nAChR. In the cholinergic anti-inflammatory pathway, acetylcholinebinding to nicotinic acetylcholine receptor subunit α7 (α7nAChR) leadsto the inhibition of the phosphorylation of inhibitor of NF-κB (IκB),the downregulation of the activation of mitogen-activated proteinkinases (MAPKs), inhibition of the release of intracellular Ca²⁺ storesand the formation of a heterodimeric protein complex with Janus kinase 1(JAK2), which activates signal transducer and activator of transcription2 (STAT3). Together, these signaling cascades lead to inhibition ofpro-inflammatory cytokine release.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed, J. Wiley &Sons (New York, N.Y. 2001); and Sambrook and Russel, Molecular Cloning:A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (ColdSpring Harbor, N.Y. 2001), provide one skilled in the art with a generalguide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used herein, “EAE” means experimental autoimmune encephalomyelitis.

As used herein, “CNS” means central nervous system.

As used herein, “MS” means multiple sclerosis.

As used herein, “PBS” means phosphate-buffered saline.

As used herein, “nicotinic receptor agent” means a compound(s) and/orcomposition that may function similarly to, or have a similar effect asnicotine, including such compound(s) that includes nicotine, or apharmaceutical equivalent, analog, derivative, or salt thereof. Thiswould include, for example, but in no way limited to, the substancesdescribed in Table 1 herein. This would include, for example, acompound(s) that may bind to and activate a nicotinic cholinergicreceptor (nAChR). This would also include, for example, a compound(s)and/or composition that modulates the signaling pathway, either upstreamor downstream, that is activated by the binding of an activating ligand(such as nicotine and acetylcholine) to heteromeric and/or homomericnAChRs. FIG. 11 herein depicts an overview of a signaling pathway of theα7 nAChR subtype. “Nicotinic receptor agent,” for example, would includeany of the compounds depicted in FIG. 11, such that they may modulatethe signaling cascade effect of nicotine or acetylcholine binding to thenAChR, including the inhibition of the phosphorylation of inhibitor ofNF-κB (IκB), the downregulation of the activation of mitogen-activatedprotein kinases (MAPKs), inhibition of the release of intracellular Ca²⁺stores and the formation of a heterodimeric protein complex with Januskinase 1 (JAK2), which activates signal transducer and activator oftranscription 2 (STAT3), and the inhibition of pro-inflammatory cytokinerelease.

As used herein, “pumps” include but is not limited to osmotic minipumps.

As disclosed herein, the inventors show that nicotine significantlyattenuates the magnitude of inflammation and autoimmune responsesagainst the myelin antigens in a mouse model of EAE. In the peripheralimmune system, nicotine inhibits the proliferation of autoreactive Tcells and alters the cytokine profile of helper T cells. In the CNS,nicotine preferentially reduced the number of migrated CD11b⁺ andCD11b⁺CD45⁺ microglial, downregulated the expression of MHC class II,CD80 and CD86 molecules on these cells. The results demonstrate thatphysiological immune mechanisms modulated by nicotine can be exploitedfor the treatment of inflammatory and autoimmune disorders in the CNS.

In one embodiment, the present invention provides a method of treating adisease and/or condition by administering a therapeutically effectiveamount of a composition comprising nicotine. In one embodiment, thepresent invention provides a method of treating a disease and/orcondition in an individual by administering a nicotinic receptor agentto the individual. In another embodiment, the disease and/or conditionis EAE. In another embodiment, the disease and/or condition is multiplesclerosis. In another embodiment, the present invention provides amethod of treating CNS inflammation and/or autoimmunity by administeringa therapeutically effective dosage of nicotinic receptor agent. Inanother embodiment, administering a therapeutically effective dosage ofnicotinic receptor agent attenuates CNS inflammation and/or autoimmuneresponsiveness to myelin antigens. In another embodiment, the nicotinicreceptor agent is administered by osmotic minipumps implantedsubcutaneously. In another embodiment, the nicotinic receptor agent isadministered intravenously by direct injection. In another embodiment,the nicotinic receptor agent includes nicotinic bitartrate. In anotherembodiment, a therapeutically effective dosage of nicotinic receptoragent is 13 mg of nicotine free base/kg/d administered to a mouse, orthe human dosage equivalent. In another embodiment, the individual is ahuman. In another embodiment, the individual is a mouse.

As further disclosed herein, the inventors have developed variousmethods of modulating the magnitude of inflammatory and autoimmuneresponse against neuroantigens by nicotine. In one embodiment, thenicotine can be used to suppress an immune response to autoantigen. Inanother embodiment, the immune response to autoantigen is in vivo. Inanother embodiment, the immune response to autoantigen is ex vivo. Inanother embodiment, the immune response to autoantigen is in vitro. Inanother embodiment, the suppression of an immune response to anautoantigen results in the treatment, amelioration and/or prevention ofan autoimmune and/or inflammatory disorder. In another embodiment, theautoimmune and/or inflammatory disorder is of the central nervoussystem.

The present invention is also directed to a kit to prepare a nicotinicreceptor agent, as well as the delivery of the nicotinic receptor agentto an individual, and may include an osmotic minipump, nicotinebitartrate, PBS, and combinations thereof. The kit is an assemblage ofmaterials or components, including at least one of the inventivecompositions. Thus, in some embodiments the kit contains a compositionincluding a therapeutically effective dosage of nicotine, as describedabove.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of delivering a therapeutically effectivedosage of nicotine to mammalian subjects, such as, but not limited to,human subjects, farm animals, domestic animals, and laboratory animals.Other embodiments, for example, are configured for preparing atherapeutically effective dosage of nicotine to mammalian subjects, suchas, but not limited to, human subjects, farm animals, domestic animals,and laboratory animals.

Instructions for use may be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to prepare a nicotinic solution and/or deliver a therapeuticallyeffective dosage of nicotinic receptor agent to treat an immunologicaldisease. Optionally, the kit also contains other useful components, suchas, diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, bandagingmaterials or other useful paraphernalia as will be readily recognized bythose of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.As used herein, the term “package” refers to a suitable solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding the individual kit components. Thus, for example, a package canbe a glass vial used to contain suitable quantities of an inventivecomposition containing a solution of nicotine or components thereof. Thepackaging material generally has an external label which indicates thecontents and/or purpose of the kit and/or its components.

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of nicotine. “Pharmaceuticallyacceptable excipient” means an excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for veterinaryuse as well as for human pharmaceutical use. Such excipients may besolid, liquid, semisolid, or, in the case of an aerosol composition,gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention may be formulated for delivery via any route ofadministration. “Route of administration” may refer to anyadministration pathway known in the art, including but not limited to anosmotic minipump, intravenous injection, aerosol, nasal, oral,transmucosal, transdermal or parenteral. “Parenteral” refers to a routeof administration that is generally associated with injection, includingintraorbital, infusion, intraarterial, intracapsular, intracardiac,intradermal, intramuscular, intraperitoneal, intrapulmonary,intraspinal, intrasternal, intrathecal, intrauterine, intravenous,subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection, or aslyophilized powders.

The nicotinic receptor agents according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier may be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it may come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The nicotinic receptor agents according to the invention can also beencapsulated, tableted or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols and water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.

The preparations of nicotinic receptor agents are made following theconventional techniques of pharmacy involving milling, mixing,granulation, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation will be in the form of a syrup,elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly p.o. or filled into a softgelatin capsule.

The nicotinic receptor agents according to the invention may bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

Typical dosages of nicotinic receptor agents can be in the rangesrecommended by the manufacturer where known therapeutic compounds areused, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. Such dosages typically can bereduced by up to about one order of magnitude in concentration or amountwithout losing the relevant biological activity. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based, forexample, on the in vitro responsiveness of the relevant primary culturedcells or histocultured tissue sample, such as the responses observed inthe appropriate animal models, as previously described. In oneembodiment, a therapeutically effective dosage of nicotinic receptoragent administered to a mouse ranges from 1 mg nicotine free base/kg/dayto 60 mg nicotine free base/kg/day, with a preferable dosage of 13 mgnicotine free base/kg/day. In another embodiment, a therapeuticallyeffective dosage of nicotinic receptor agent administered to a humanranges from nicotine plasma levels of 1 ng/mL to 100 ng/mL, with apreferable dosage of 30 ng/mL. In another embodiment, a therapeuticallyeffective dosage of nicotinic receptor agent administered to a human asa single dose ranges from 0.1 mg to 5 mg, with 1 mg the preferred singledose. In another embodiment, a therapeutically effective dosage ofnicotinic receptor agent administered to a human ranges from 0.015mg/kg/day to 0.6 mg/kg/day, with a preferable dosage of 0.3 mg/kg/day.

As described herein, various embodiments of the invention include theattenuation of CNS inflammation and autoimmune responsiveness toantigens. As readily apparent to one of skill in the art, the inventionmay be applied to any number of CNS conditions and diseases, includingstroke, trauma, Alzheimer's disease, Parkinson's disease, epilepsy,encephalomyelitis and MS.

Additionally, as would be readily apparent to one of skill in the art,any number of methods and devices commercially available may be used,either alone or in conjunction with various embodiments describedherein, to deliver a therapeutically effective dosage of nicotinicreceptor agent, and the invention is in no way limited to intravenousdelivery by injection or through subcutaneous implantation of osmoticminipumps. This would include, but not limited to, nasal sprays, oralnicotine, nicotine patches, nicotine gum, and nicotine inhalers.

Additionally, as readily apparent to one of skill in the art, there areany number of nicotinic compounds, either alone or in conjunction withvarious embodiments described herein, that could be used for thedelivery of a therapeutically effective dosage of nicotinic receptoragent, and the invention is in no way limited to use of nicotinebitartrate. This would include, but not limited to, nicotine mimetics.Similarly, nicotine exposure-induced decreases in nicotinic receptorfunction may also have a therapeutic effect. This would include, but notlimited to, agents or their analogs such as bupropion or mecamylamine,and non-competitive antagonists of nicotinic receptor subtypes orcompetitive inhibitors such as methyllycaconitine.

Similarly, as readily apparent to one of skill in the art, the inventionmay also be used in the form of adjuvant therapies. In one embodiment, adisease may be treated by administering a therepeutically effectivedosage of a nicotinic receptor agent in conjunction with administering athereapeutically effective dosage of a composition, where a conditionassociated with the disease is treated by administering the nicotinicreceptor agent, and the disease itself is treated by administering thecomposition. In one embodiment, the condition associated with thedisease is CNS inflammation. In another embodiment, the disease is aneurodegenerative disease. In another embodiment, the disease is stroke,trauma, Alzheimer's disease, Parkinson's disease, and/or epilepsy. Inanother embodiment, the condition associated with the disease is anautoimmune effect. In another embodiment, the disease is HIV/AIDS,lupus, encephalomyelitis and/or MS.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Generally

The expression of nicotinic acetylcholine receptors in the non-neuronalcells, microglia and astrocytes, is evidence that the nicotinic receptoractivity is involved in immune responses within the central nervoussystem (CNS). The inventors show that nicotine significantly attenuatesthe magnitude of inflammation and autoimmune responses against themyelin antigens in a mouse model of experimental autoimmuneencephalomyelitis (EAE). In the peripheral immune system, nicotineinhibits the proliferation of autoreactive T cells and alters thecytokine profile of helper T cells. In the CNS, nicotine preferentiallyreduced the number of migrated CD11b⁺ and CD11b⁺CD45⁺ microglia, anddownregulated the expression of MHC class II, CD80 and CD86 molecules onthese cells. Results described herein show that physiological immunemechanisms modulated by nicotine can be exploited for the treatment ofinflammatory and autoimmune disorders in the CNS.

Example 2 Mice

B6 (H-2) mice purchased from Taconic (Germantown, N.Y., USA) were housedin pathogen-free animal facilities of the Barrow Neurological Institute,Phoenix, Arizona. The female mice used were 7 to 8 weeks of age at theexperiment's inception. Experiments were conducted in accordance withinstitutional guidelines.

Example 3 Antigens

The murine MOG₃₅₋₅₅ peptide (SEQ. ID. NO.: 1) and proteolipid protein(PLP)₁₃₉₋₁₅₁ peptide (SEQ. ID. NO.: 2) were synthesized by Bio SynthesisInc. (Lewisville, Tex., USA).

Example 4 Induction of Acute EAE and Adoptive Transfer of EAE

To induce acute EAE, B6 mice were injected subcutaneously (s.c.) in thehind flank with 200 μs of MOG₃₅₋₅₅ peptide in complete Freund's adjuvant(CFA) (Difco, Detroit, Mich., USA) containing 500 μg of mycobacteriumtuberculosis. On the day of immunization and 2 days afterward, the micewere injected intraperitoneally (i.p.) with 200 ng pertussis toxin (ListBiologic, Campbell, Calif., USA).

For adoptive transfers of EAE, lymph node cells were obtained from thesemice on day 8 after immunization and cultured at a density of 2×10⁶/mlin Click's EHAA medium supplemented with 15% FCS, 20 ng/ml recombinantIL-12, and 50 μg/ml MOG₃₅₋₅₅ peptide. After 4 days of culture, cellswere harvested, and 3×10⁷ viable cells were injected i.p. into eachrecipient mouse irradiated (350 rads) 1 h earlier. For both actively andpassively induced EAE, the mice were observed daily for clinical signsof disease and scored on an arbitrary scale of 0 to 5, with gradationsof 0.5 for intermediate scores: 0, no clinical signs; 1, flaccid tail;2, hind limb weakness or abnormal gait; 3, complete hind limb paralysis;4, complete hind limb paralysis with forelimb weakness or paralysis; 5,moribund or deceased.

Example 5 Nicotine Treatment

Nicotine bitartrate was purchased from Sigma (St. Louis, Mo., USA). A100 mg/ml solution of nicotine bitartrate in phosphate-buffered saline(PBS) was freshly prepared 24 h before pump implantation. The solutionwas loaded into Alzet® osmotic minipumps (model 1007D, DurectCorporation, Cupertino, Calif., USA); the delivery rate is 12 μl/d or0.39 mg/mouse/d. For a mouse approximately 30 gm, this equates to 13 mgof nicotine free base/kg/d or approximately 0.54 mg of nicotine freebase/kg/hr. These minipumps were then implanted s.c. into the mice tocontinuously deliver nicotine or PBS for 7 days. Control mice receivedPBS via minipumps or direct injections, both of which produced identicalresults, as described herein.

To determine the ability of nicotine pretreatment to prevent anautoimmune response, mice received 13 mg/kg nicotine or PBS daily for atotal of 7 doses starting on the day of MOG immunization to induce EAE(day 0) or 7 days earlier. To evaluate the effect of nicotine on anongoing autoimmune response, mice received 13 mg/kg nicotine or PBSdaily for a total of 7 doses on day 7 after EAE induction. For adoptivetransfer of EAE, lymphocytes were isolated from mice that were treatedwith nicotine at a dose of 13 mg/kg or PBS daily for a total of 7 dosesat the time of MOG immunization.

Example 6 Preparation of Tissues and Histological Staining

Mice were anesthetized with pentobarbital and perfused by intracardiacpuncture with 50 ml of cold PBS. Spinal cords were removed and fixed in10% formalin/PBS. Paraffin-embedded longitudinal sections were preparedand stained for hematoxylin and eosin (H&E), luxol fast blue (LFB,myelin staining) and Biechowsky silver (axon staining). The percentagesof spinal cord inflammation, demyelination and axonal damage per mousewere calculated by first determining the total white matter area for allspinal cord sections by manually tracing the regions. Next, the areas ofspinal cord inflammation, demyelination and axonal damage weredetermined by manually tracing each section. Pathological changes ineach spinal cord were scored as follows: 0 no changes; 1 focal areainvolvement; 2 <5% of total myelin area involved; 3 5-10% of totalmyelin area involved; 4 10-20% involved area; 5 >20% of total myelinarea involved (Bai, X. F., et al., J Exp Med 200:447-458).

Example 7 T Cell Proliferation Assays

Spleen mononuclear cells were suspended in culture medium containingDulbecco's modification of Eagle's medium (Gibco, Paisley, UK)supplemented with 1% (v/v) minimum essential medium (Gibco), 2 mMglutamine (Flow Laboratory, Irvine, Calif., USA), 50 IU/ml penicillin,50 mg/ml streptomycin and 10% (v/v) FCS (both from Gibco). Spleenmononuclear cells (4×10⁵ cells/well) in 200 μl of culture medium wereplaced in 96-well round-bottom microtiter plates (Nunc, Copenhagen,Denmark). Ten μl of MOG₃₅₋₅₅ peptide (10 μg/ml), PLP₁₃₉₋₁₅₁ peptide (10μg/ml) or Con A (5 μg/ml) (Sigma-Aldrich, St Louis, Mo., USA) were thenadded into triplicate wells. After 3 days of incubation, the cells werepulsed for 18 h with 10 μl aliquots containing 1 μCi of³H-methylthymidine (sp. act. of 42 Ci/mmo; MP Biomedicals, Irvine,Calif., USA). Cells were harvested onto glass fiber filters, andthymidine incorporation was then measured. The results were expressed ascounts per minute (cpm).

Single cell suspensions (4×10⁷ cells) were prepared and labeled with 0.5μM CFSE at 37° C. for 10 min. Cells with or without CFSE were incubatedat 37° C. for 3 days in round-bottom plates (2×10⁶ cells/well) with orwithout antigens (MOG 10 μg/ml). After harvesting, cells were stainedfor surface markers with fluorochrome-conjugated monoclonal antibodiesincluding anti-CD3-PE/Cy5 (17A2), anti-CD4-APC/Cy7 (GK1.5), andanti-CD8α-PE/Cy7 (53-6.7) (BD Bioscience, San Diego, Calif., USA).Isotype-matched negative monoclonal antibodies were used as controls.

Example 8 Cell Viability and Apoptosis Assay

Cell viability was assessed by trypan blue dye exclusion. For detectionof cell apoptosis, the spleen mononuclear cell suspensions werecollected from PBS and nicotine-treated mice on day 11 (peak stage).Single cell suspensions were washed in PBS and resuspended in bindingbuffer containing annexin V-FITC and propidium iodide (PI) (both from BDBiosciences) for 20 min at room temperature. The samples were analyzedon a FACSAria using Diva.

Example 9 Spleen and CNS Cell Isolation and Flow Cytometric Analysis

Spleen mononuclear cell suspensions were collected from PBS andnicotine-treated mice on day 11 (peak stage). Single cell suspensionswere prepared and stained with fluorescently labeled antibodies to mouseantigens. Antibodies were directly labeled with one of the followingfluorescent tags: FITC, PE, APC, PE-Cy5, PE-Cy-7, CD25 (PC61.5),APC-Cy7; CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), NK1.1 (PK136), CD11b(M1/70), CD11c (HL3), CD19 (1D3), CD80 (16-10A1), CD86 (GL1), MHC classII (M5/114.15), and TCRβ (H57-597). Intracellular Foxp3 (FJK-16s) cellswere stained as the manufacture instructed (eBioscience, San Diego,Calif., USA). Appropriate isotype controls were always included. Allsamples were analyzed on a FACSAria using Diva. The absolute number of aparticular cell subset was calculated by counting the mean of totalmononuclear cells isolated per mouse spleen and multiplying by thepercentage of those cells acquired by FACSAria flow cytometry.

For CNS cell isolates, at day 11 after EAE induction (peak stage), micewere sacrificed and perfused with PBS through the left heart ventricleto eliminate contaminating blood cells in the CNS. The CNS mononuclearcells were then isolated from five to six mice using Percoll gradientsand stained for cell surface markers. Antibodies were directly labeledand analyzed as done for splenocytes. The absolute number of aparticular cell subset was calculated by counting the total number ofmononuclear cells isolated from the CNS of individual mice andmultiplying by the percentage of those cells acquired by FACSAria flowcytometry (Bai, X. F., et al., J Exp Med 200:447-458).

Example 10 Cytokine Quantification

Single cell suspensions (4×10⁷ cells) were prepared and incubated at 37°C. for 3 days in round-bottom plates (2×10⁶ cells/well) with or withoutantigens (MOG 10 μg/ml, PLP 10 g/ml or Con A 2.5 μg/ml), and stimulatedwith PMA (20 ng/ml)/ionomycin (1 μg/ml)/brefeldin A (5 μg/ml) foranother 5 h at 37° C. After harvesting, cells were stained for surfacemarkers with fluorochrome-conjugated monoclonal antibodies includinganti-CD3-PE/Cy5 (17A2), anti-CD4-APC/Cy7 (GK1.5), and anti-CD8α-PE/Cy7(53-6.7) (BD Bioscience). Isotype-matched negative monoclonal antibodieswere used as controls. For intracellular cytokine staining, afterfixation and permeabilization with Cytofix/Cytoperm kit (BD Bioscience),anti-IL-4, anti-IL-10 and anti-IL-17 monoclonal antibody conjugated withAlexa 647 were used. All samples were analyzed on a FACSAria using Diva.To determine the percentage of cells producing cytokine, the valueobtained with the isotype control was subtracted from that with specificantibody. For cytokine induction, supernatants were collected 3 daysafter in vitro boosting. IFN-γ, IL-10, IL-2 and TGF-β were measured byoptEIA kits (PharMingen and eBioscience).

Example 11 Quantification of MOG-Reactive Antibodies

MOG-reactive antibodies were quantified by enzyme-linked immunoabsorbentassay (ELISA). Briefly, microtiter plates (Corning Glass Works, Corning,N.Y., USA) were coated with 100 μl/well of murine MOG₃₅₋₅₅ (10 μg/ml) at4° C. overnight. After blocking with 10% fetal bovine serum, serumsamples (day 11, peak stage after immunization) were added and incubatedat 4° C. overnight. Plates were then incubated for 2 h with biotinylatedrabbit anti-mouse IgG, IgG1, IgG2a, IgA, IgG3 and IgG2b (Invitrogen,Carlsbad, Calif., USA), followed by alkaline phosphatase-conjugated ABCreagent (Dakopatts; R&D systems, Minneapolis, Minn., USA). The color wasdeveloped with p-nitrophenyl-phosphate. Results were expressed asoptical density (OD) at 450 nm.

Example 12 Statistical Analysis

Differences between groups were evaluated by ANOVA. The Fisher's exacttest and Mann-Whitney's U-test were applied to analyze disease incidenceand severity, respectively.

Example 13 Nicotinic Attenuation of Actively Induced—and AdoptivelyTransferred—EAE

To determine how nicotine might influence the course of EAE, B6 micewere infused with PBS or nicotine at a dose of 13 mg/kg for 7 days byusing an implantable minipump. The decision on dosage and route ofadministration was based on recently published guidelines for testingthe effects of nicotine in vivo (Matta, S. G., et al.,Psychopharmacology (Berl) 190:269-319). The dosage applied in the studyis estimated close to physiological dosage of nicotine.

A single injection of MOG₃₅₋₅₅ peptide together with CFA and pertusistoxin induced moderate to severe EAE (mean maximal clinical score4.25±0.80, mean clinical score 2.99±0.67) in the majority of B6 micetested. The average time of disease onset was 7.6±0.53 dayspost-immunization (p.i.). Ascending paralysis ensued at about 9 to 14days, followed by some degree of recovery with a residual neurologicaldeficit at the experiment's termination (mean clinical score 2.75±0.25)at day 30 p.i. In contrast, mice receiving nicotine prior to or the dayof disease induction had a delayed onset of EAE, which was not apparentuntil day 10 p.i. Nicotine-treated groups developed EAE with meanmaximal clinical scores of 3.13±0.25 (nicotine treatment group, startingday 0 after immunization) and 3.17±0.78 (pre-treatment group, startingday −7 after immunization). The latter's disease was significantly lesssevere than that of control mice given PBS, and the mean day of diseaseonset after nicotine treatment was clearly later than that of PBScontrols (p<0.01). Nicotine treatment before or at the time of diseaseinduction showed similar peak clinical scores and delayed onset of EAE(p<0.05,p<0.01, respectively, when compared with that of controlanimals). The disease of nicotine-treated mice was relatively mild, atmaximum 3.13±0.25 vs. 4.25±0.80 (p<0.01). The majority of animalrecovered by day 21 (nicotine pre-treated group) or 24 (nicotine-treatedgroup) with a mild motor deficit remaining (mean clinical score0.83±0.26, 0.85±0.25 vs. 2.75±0.25,p<0.01, respectively). Thus, nicotineexposure preceding or simultaneous with disease induction significantlydelayed its onset, attenuated its severity and promoted the recovery. Todetermine whether nicotine is capable of altering the expression of EAEwhen myelin-reactive T cells become activated and early signs of diseasebegin to manifest, groups of mice were immunized with MOG and CFA. Atday 7 p.i. when EAE signs began (flaccid tail, hind limb weakness,etc.), all immunized mice were randomly separated into two groups: onereceived PBS and the other nicotine. Compared with mice receiving PBS(maximal clinical score 3.3±0.25, mean clinical score 1.89±0.43),recipients of nicotine had significantly milder EAE (FIG. 1C, p<0.05 andp<0.01, respectively). Furthermore, nicotine improved their recoveryfrom motor weakness over that of control mice (p<0.01, mean clinicalscore 0.69±0.43 at the termination of experiments). Therefore, theresults clearly demonstrate that nicotine halted the progression of EAEwhen myelin-reactive T cells were activated as signs of clinical EAEbecame visible.

EAE is a T-cell mediated disease in that it can be produced inrecipients of encephalitogenic T cell transfers (McRae, B. L., et al., JNeuroimmunol 38:229-240). Thus, it was important to determine thecapacity of myelin-reactive T cells from nicotine-treated mice to induceEAE. Accordingly, when the inventors performed adoptive transfers of Tcells from nicotine-treated animals, as described herein, a milder formof EAE resulted than that from T cells of control mice (maximal clinicalscore 2.5±0.82 vs. 5.0±0.25 in nicotine-treated vs. control mice,respectively, p<0.001; mean clinical score 4.22±0.36 vs1.0±0.15,p<0.001). Collectively, the results have shown that nicotineattenuated EAE when present before or after T cell activation, and thatT cells from nicotine-treated mice significantly lowered the capacity toproduce EAE in recipients of such transfers.

Example 14 Nicotine Ameliorates CNS Inflammation, Demyelination andAxonal Damage

Pronounced cellular infiltration, demyelination and axonal damage arepathological hallmarks of EAE and MS. To evaluate whether nicotine canalter these pathological changes, the inventors examined spinal cordsfrom mice with ongoing EAE. In the white matter of these tissues fromcontrol mice, H&E and LFB staining revealed marked multifocal andlymphohistiocytic inflammation that was both perivascular and diffuse.Myelin loss was widespread, especially around inflamed areas. In sharpcontrast, most sections from nicotine-treated mice had few infiltratingcells, and myelin sheets were largely preserved. Furthermore, axonaldamage was clearly present in the sub-meningeal areas of PBS controlmice, whereas axons from nicotine-treated mice were scarcely affectedand then only in regions immediately surrounding foci of inflammation.Quantification showed that inflammation, demyelination and axonaldegeneration were significantly greater in PBS-treated compared tonicotine-treated mice (p<0.01). Therefore, nicotine was responsible forprotecting the CNS from inflammation, demyelination and axonal damageotherwise caused by EAE.

Example 15 Nicotine Alters the Peripheral Lymphocyte SubpopulationDuring EAE

Nicotine promotes the development of T and B cells during ontogeny(Middlebrook, A. J., et al., J Immunol 169:2915-2924; Skok, M., et al.,J Neuroimmunol 171:86-98; Skok, M., et al., Eur Pharmacol 517:246-251)and has pro- and anti-apoptotic activities for mature cells includinglymphocytes (Zeidler, R., et al., Apoptosis 12:1927-1943). To addresswhether nicotine might alter the homeostasis of lymphocytes during anautoimmune response to MOG, the inventors quantified various lymphocytesubpopulations. For this and subsequent sections, cells from mice givennicotine at the day of MOG immunization (day 0) were used forimmunological analysis. Results obtained from other time points weresimilar. Compared to control EAE mice, nicotine-treated mice had variousdegrees of reductions in the percentages and numbers of CD3⁺, CD4⁺ andCD8⁺ T cells as well as CD19⁺CD3⁻ B cells among splenocytes sampled onday 11 p.i. (p<0.05). With respect to NK (CD3⁻NK1.1⁺ cells) cells, NKTcells and CD4⁺CD25⁺ regulatory T cells, however, the values did notdiffer significantly between the nicotine-treated mice and PBS-treatedmice at this time point. Interestingly, the expression of Foxp3 wasclearly augmented in the nicotine-treated mice. Although the numbers andpercentages of CD11c⁺ and CD11B⁺ cells were not dramatically altered bynicotine treatment, reductions in the expression of MHC class II, CD80and CD86 were notable on these cells. Similar data were also obtainedfrom assessments of lymph nodes and blood at several other time pointsduring the course of EAE.

Example 16 Nicotine Inhibits Autoreactive T cell Expansion

To address how nicotine affected expansion of MOG₃₅₋₅₅-specific T cellresponses in PBS- vs. nicotine-treated EAE mice, mononuclear cells wereisolated from their spleens, and the proliferation of T cells inresponse to myelin antigens was quantified using ³H incorporation aswell as CFSE assays. Splenocytes from PBS-treated controls andnicotine-treated groups mounted significant proliferative responses toMOG. However, the nicotine recipients developed far fewer T cells inresponse to the immunizing MOG antigen than did the PBS recipients(p<0.05 vs. PBS). Similarly, a reduced proliferation of CD3⁺, CD4⁺ andCD8⁺ T cells in response to MOG was also recorded in nicotine-treatedmice by using the CFSE assay. Next, annexin V and PI double stainingwere used to determine the effect of nicotine on T cell apoptosis anddeath. The results demonstrated that nicotine did not induce T cellapoptosis, at least at the current dose. Thus, the reduced numbers ofseveral lymphocyte subpopulations recorded herein were the likely resultof their decline in proliferation after nicotine exposure.

Example 17 Nicotine Alters Th Cell Cytokine Profile

To further characterize autoreactive T cells, the inventors investigatedthe effect of nicotine on cytokine production by Th cells. The inventorsfirst detected cytokines in the supernatants of splenocytes culturedwith MOG₃₅₋₅₅. There was a significant decrease of IFN-γ production inthe nicotine-treated animals compared to controls. Further, the majorcontributors to this reduction appeared to be CD8⁺ rather than CD4⁺cells. Similarly, the production of IL-2 decreased in thenicotine-treated animal. In contrast, production of IL-10 and TGF-β1(p<0.05,p<0.01, respectively) was significantly augmented by nicotinetreatment. Collectively, nicotine inhibited the myelin antigen-inducedproduction of IFN-γ and IL-2 yet augmented the production of IL-10 andTGF-β.

Example 18 Nicotine Alters Autoantibody Isotypes

Autoantigen responses can reflect T cell help; for example, in mice,IFN-γ is believed to drive the IgG2b response, whereas the IgG1 responseis driven by Th2 cells. The inventors therefore used an ELISA to measureMOG-specific antibodies in PBS- and nicotine-treated mice. Consistentwith the Th cytokine profile, IgG2b production in nicotine-treated micewas profoundly impaired, whereas synthesis of IgG1 and IgA was markedlyincreased compared with that in control mice.

Example 19 Nicotine Alters Lymphocyte Subpopulations in the ENS DuringEAE

Histopathological studies revealed that less inflammatory infiltrationoccurs in CNS tissues of nicotine-treated animal than those ofPBS-treated controls. To document nicotine's alteration of the cellularspectrum in the CNS during EAE, the inventors analyzed inflammatorycells that migrated into the CNS as well as residual microglia byisolating CNS mononuclear cells. As expected, T cells (CD3⁺, CD4⁺, CD8⁺)and B cells (CD3⁻CD19⁺) were abundant in CNS sections from PBS-treatedEAE mice, but their numbers were drastically reduced in thenicotine-treated mice.

Next, the inventors characterized APCs in the CNS during EAE. Whenquantifying the macrophage/microglia (CD11b⁺ cells) and dendritic cell(CD11c⁺ cells) populations in the CNS, the inventors found lowerpercentages and absolute numbers of CD11b⁺ and CD11c⁺ cells,particularly CD11b⁺ cells, in the nicotine-treated mice. Further, theexpression of MHC class II, CD80 and CD86 was reduced on the latter'sCD11c and CD11b cells. It is important to note that, compared to resultsin the periphery, reductions in expression of MHC class II, CD80 andCD86 were much more dramatic on CD11b⁺ cells of the CNS from thenicotine-treated animals. The unparalleled alteration in CD11b⁺ cellsfrom the CNS compared to the periphery implies that nicotine might beresponsible for quite different effects in these two anatomicalcompartments.

Example 20 Conclusions

Expression of nicotinic acetylcholine receptors on non neuronal cellssuch as APCs underscores the idea that they have functions well beyondneurotransmission. Previous studies indicate that nicotine exertsanti-inflammatory and immune modulatory effects in vivo (Sopori, M., NatRev Immunol 2:372-377). However, its role in CNS inflammation andautoimmune responses was not known. Using the EAE model, the inventorshave now demonstrated that nicotine can dramatically attenuate theinfiltration of inflammatory cells into the CNS as well as the relateddestruction of myelin and axons. The results thus reveal new aspects ofthe way in which nicotine functions as a potent immune modulator withinthe CNS and highlight the importance of understanding interactionsbetween the nervous system and immune system when seeking means toameliorate CNS inflammatory disorders.

Example 21 Nicotine Dosing in Mice and Humans

Nicotine bitartrate was delivered using osmotic minipumps implantedsubcutaneously in mice (100 mg/ml; molecular mass of 498 daltons orgrams/mole as the dihydrate; 12 μl/d; equates to 0.39 mg of nicotinefree base per mouse per day). For a ˜30 gm mouse, this equates to ˜13 mgof nicotine free base/kg/d or ˜0.54 mg of nicotine free base/kg/hr.Published literature indicates that plasma nicotine levels in mice are˜200 ng/ml (˜1.2 μM) after infusion of 4 mg/kg/hr of drug and ˜45 ng/ml(˜280 nM) after infusion at ˜0.5 mg/kg/hr. For comparison, human heavysmokers have plasma nicotine levels of 15-38 ng/ml (˜90-230 nM). Thus,nicotine levels in plasma of mice used in the studies are comparable tothose in the plasma of human smokers.

Example 22 Table 1—Nicotinic Analogs and/or Derivatives

Table 1 lists various examples of nicotinic receptor agents, as well aspossible sources in the literature that describe the correspondingsubstance in greater detail. Nicotinic receptor agents include nicotine,pharmaceutical equivalents, analogs, derivatives, and salts thereof,that may be used in conjunction with various embodiments describedherein. In one embodiment, a therapeutically effective dosage of asubstance described in Table 1 below may be administered to a subject totreat a degenerative disease of the central nervous system (CNS).Additionally, various examples of nictonic receptor agents are alsodescribed in references Horenstein, et al., Mol Pharmacol 74:1496-511,2008, and Arneric, et al., Biochemical Pharmacology 74 (2007) 1092-1101,incorporated by reference herein.

TABLE 1 Substance Source cytisine Acta Polon Pharm 29(5): 490; 1972Biokhimiia 43(7): 1150; 1978 nicotine polacrilex Compr Ther 1987; 13(3):32 nornicotine Environ N-Nitroso Cpds Anal Forum W1 121k No. 14: 227;1976 J Org Chem 41(21): 3438; 1976 nicotine 1-N-oxide Appl EnvironMicrobiol 1979; 38(5): 836 metanicotine J Pharmacol Exp Ther 196(3):685; 1976 nicotine imine Adv Exp Med Biol 1982; 136B: 1121 nicotineN-glucuronide J Chromatogr 1993 Nov. 17; 621(1): 49-53N-methylnicotinium J Chromatogr 1985; 347(3): 405 N-n-decylnicotinium BrJ Pharmacol 1999 November; 128(6): 1291-9 5′-cyanonicotine J Biol Chem248(8): 2796; 1973 3,4-dihydrometanicotine Klin Wochenschr 1984; 62suppl 2: 92 N′-methylnicotinium Pharmacol Biochem Behav 1991; 38(4): 843M-octanoylnornicotine Endocr Res 1991; 17(3-4): 409-192,3,3a,4,5,9b-hexahydro-1- J Med Chem 1993 Oct. 29; 36(22): 3381-5methyl-1H-pyrrolo(3,2- h)isoquinoline 5-isothiocyanonicotine BiochemPharmacol 1994 Jun. 1; 47(11): 1965-7 5-iodonicotine Biol Pharm Bull1995 November; 18(11): 1463-6 5′-hydroxycotinine-N-oxide Xenobiotica1999 August; 29(8): 793-801 homoazanicotine J Med Chem 2002 Oct. 10;45(21): 4724-31 nicotine monomethiodide Naunyn Schmiedebergs ArchPharmacol 22(282): R69; 1974 N-azido-2-nitrophenylnornicotine Ann NYAcad Sci 1980; 346: 419 N-methylnornicotinium Drug Metab Dispos 1985;13(3): 348 nicotinium molybdophosphate resin Bull Environ Contam Toxicol1986; 36(6): 924 N-methyl-N′-oxonicotinium Drug Metab Dispos 1986;14(5): 574 N′-propylnornicotine J Chromatogr 1990; 525(2): 349pseudooxynicotine Ann NY Acad Sci 1993 May 28; 686: 213-284′-methytnicotine J Med Chem 1994 Oct. 14; 37(21): 3542-535-fluoronicotine Neurochem Res 1995 September; 20(9): 1089-94K(s-nic)5(Ga2(N,N′-bis-(2,3- Inorg Chem 2001 May 7; 40(10): 2216-7dihydroxybenzoyl)-1,4- phenylenediamine)3) 5-methoxynicotine Eur JPharmacol 2002 Jan. 25; 435(2-3): 171-801-benzyl-4-phenylnicotinamidinium J Am Chem Soc 2002 Aug. 7; 124(31):9181-8 6-n-propylnicotine Bioorg Med Chem Lett 2002 Oct. 21; 12(20):3005-7 SIB1663 Brain Res 2004 Apr. 2; 1003(1-2): 42-53 6-hydroxynicotineNucleic Acids Res 2005; 33(12) (dup#1): e107 N-methyl-nicotine BiolReprod. 2005 March; 72(3): 628-32 6-(2-phenylethyl)nicotine Bioorg MedChem Lett 2005 Jul. 1; 15(13): 3237-40 N′-formylnornicotinePhytochemistry 2005 October; 66(20): 2432-40 N-n-octylnicotinium AAPS J2005; 7(1): E201-17 N-(n-oct-3-enyl)nicotinium AAPS J 2005; 7(1):E201-17 N-(n-dec-9-enyl)nicotinium AAPS J 2005; 7(1): E201-175′-acetoxy-N′-nitrosonornicotine Chem Res Toxicol 2006 March; 19(3):426-35 4-hydroxynicotine Org Lett. 2005 Oct. 27; 7(22): 5059-624-(dimethylphenylsilyl)nicotine Org Lett. 2005 Oct. 27; 7(22): 5059-62N′-carbomethoxynornicotine J Nat Can Inst 54(5): 1238; 1975N-methylnicoton Arch Pharm (Weinheim) 309(3): 197; 1976

While the description above refers to particular embodiments of thepresent invention, it should be readily apparent to people of ordinaryskill in the art that a number of modifications may be made withoutdeparting from the spirit thereof. The presently disclosed embodimentsare, therefore, to be considered in all respects as illustrative and notrestrictive.

Various embodiments of the invention are described above in the DetailedDescription. While these descriptions directly describe the aboveembodiments, it is understood that those skilled in the art may conceivemodifications and/or variations to the specific embodiments shown anddescribed herein. Any such modifications or variations that fall withinthe purview of this description are intended to be included therein aswell. Unless specifically noted, it is the intention of the inventorthat the words and phrases in the specification and claims be given theordinary and accustomed meanings to those of ordinary skill in theapplicable art(s).

The foregoing description of various embodiments of the invention knownto the applicant at this time of filing the application has beenpresented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit

1. A method of treating inflammation in a central nervous system of asubject, the method comprising the steps of: administering to thesubject a therapeutically effective dose of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises anicotinic receptor agent.
 2. The method of claim 1, wherein theinflammation in the central nervous system is caused by an immuneresponse to myelin.
 3. The method of claim 1, wherein the nicotinicreceptor agent is selected from the list of compounds in Table
 1. 4. Themethod of claim 1, wherein administration of the therapeuticallyeffective dose of the pharmaceutical composition alters the function ofT cells, B cells, microglia, monocytes, macrophages, natural killercells, eosinophils, dendritic cells or other immune system cell typesfound in the central nervous system.
 5. The method of claim 1, whereinthe therapeutically effective dosage from 0.1 mg nicotine free base/kgto 60 mg nicotine free base/kg.
 6. The method of claim 1, wherein theinflammation in the central nervous system is caused by at least one ofthe following: cancer, Alzheimer's disease, and presence of amyloid-betaplaques.
 7. The method of claim 1, wherein the therapeutically effectivedosage of the pharmaceutical composition provides for nicotine plasmalevels of 1 ng/mL to 100 ng/mL.
 8. The method of claim 1, wherein theinflammation in the central nervous system is caused by trauma.
 9. Amethod of treating post-stroke inflammation in a central nervous systemof a subject, the method comprising the steps of: administering to thesubject a therapeutically effective dose of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises anicotinic receptor agent, wherein the subject is experiencingpost-stroke inflammation in the central nervous system.
 10. The methodof claim 9, wherein the nicotinic receptor agent is selected from thelist of compounds in Table
 1. 11. The method of claim 9, wherein thenicotinic receptor agent comprises nicotine bitartrate or nicotine. 12.The method of claim 9, wherein the pharmaceutical composition isadministered to the subject orally, transdermally, nasally, via a gum,via a patch, via an aerosol, via a nasal spray, or via an inhaler. 13.The method of claim 9, wherein a therapeutically effective dose of thepharmaceutical composition is administered to the subject at least onetime per day for at least 7 days.
 14. The method of claim 9, wherein thenicotinic receptor agent is an agonist of nicotinic acetylcholinereceptor containing an α7 subunit.
 15. The method of claim 9, whereinthe pharmaceutical composition comprises a pharmaceutically acceptablecarrier.
 16. A method of treating autoimmunity in a central nervoussystem of a subject, the method comprising the steps of: administeringto the subject a therapeutically effective dose of a pharmaceuticalcomposition, wherein the pharmaceutical composition comprises anicotinic receptor agent.
 17. The method of claim 16, wherein theautoimmunity in the central nervous system is caused by an immuneresponse to myelin.
 18. The method of claim 16, wherein administrationof the therapeutically effective dose of the pharmaceutical compositioninhibits proliferation of autoreactive T cells or B cells.
 19. Themethod of claim 16, wherein the autoimmunity in the central nervoussystem is caused by trauma.
 20. The method of claim 16, wherein thenicotinic receptor agent is selected from the list of compounds in Table1.